Oxazolidinone for treatment of infections with Mycobacterium tuberculosis

ABSTRACT

The present invention is a method of treating or preventing  Mycobacterium tuberculosis  infection in a subject by administering to the subject an effective amount of oxazolidinone, specifically (N-(((S)-3-(dibenzo[b,e][1,4]dioxin-7-yl)-2-oxooxazolidin-5-yl)methyl)acetamide) or a pharmaceutically acceptable salt, solvate, or stereoisomer thereof.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a 35 U.S.C. § 371 U.S. national, entry ofInternational Application PCT/US2017/018248, having an internationalfiling date of Feb. 17, 2017, which claims the benefit of U.S.Provisional Application No. 62/296,160, filed Feb. 17, 2016, the contentof each of the aforementioned applications is herein incorporated byreference in their entirety.

STATEMENT OF GOVERNMENTAL INTEREST

This invention was made with government support under grant no.OD008459, awarded by the National Institutes of Health. The governmenthas certain rights in the invention.

BACKGROUND OF THE INVENTION

In 2008, the Infectious Diseases Society of America (IDSA) made one ormore ominous declaration(s) based on the ongoing explosion ofantibiotic-resistant infections that continues to plague global healthcare. Equally alarming is the decline in the research and development ofnew antibiotics to deal with this threat of antibiotic resistantorganisms. In 2009, IDSA sounded another alarm by emphasizing theirconcern that the infrastructure for discovering and developing newantibacterials continues to stagnate, thereby risking the futurepipeline of antibacterial drugs. Of major concern are the community andhealth care associated infections with bacterial pathogens Enterococcusfaecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacterbaumannii, Pseudomonas aeruginosa, and Enterobacter species (or commonlyabbreviated as ESKAPE) that are increasingly becoming resistant to drugsavailable to treat them.

The synthesis of a novel oxazolidinone, T145(N-MS)-3-(dibenzo[b,e][1,4]dioxin-7-yl)-2-oxooxazolidin-5-yl)methyl)acetamide)that exhibits antimicrobial potency against methicillin-sensitiveStaphylococcus aureus was described (J.T. Bioorganic & MedicinalChemistry 2008, 16, 2651). Linezolid, an oxazodilinone, initiallylabeled as U-100766, is a Food and Drug Administration approvedantibacterial that is used for treatment of a wide range of bacterialpathogens. Oxazolidinones have a common 2-oxazolidone ring and inhibitinitiation of protein synthesis by preventing formation of ribosome andN-formylmethionyl-tRNA complex. Resistance to oxazolidinones generallyresult from mutation in 23S rRNA, which supports its mechanism ofaction, or due to activity of efflux pumps.

Although the World Health Organization declared tuberculosis a globalhealth threat more than 20 years ago, this year it increased itsestimate of death from this disease to 1.5 million making it the numberone bacterial infectious disease in the world. Although tuberculosis isan ancient disease and Mycobacterium tuberculosis, its etiologicalagent, was discovered in 1882, it continues to be a major globalinfectious disease. Emergence of multiple- and extensively-drugresistant strains of Mycobacterium tuberculosis in many regions of theworld clearly demonstrates that efforts to innovate new agents againstthis pathogen have been inadequate.

SUMMARY OF THE INVENTION

One embodiment of the present invention is a method of treating orpreventing Mycobacterium tuberculosis infection in a subject, an animalpreferably a human, by administering to the subject an effective amountof a pharmaceutical composition comprising:

T145:(N-(((S)-3-(dibenzo[b,e][1,4]dioxin-7-yl)-2-oxooxazolidin-5-yl)methyl)acetamide)

or a pharmaceutically acceptable salt, solvate, or stereoisomer thereof.Additionally one or more antimicrobial compounds may be administered.

Another embodiment of the present invention is a method for theselection and isolation of bacteria not sensitive to(N-(((S)-3-(dibenzo[b,e][1,4]dioxin-7-yl)-2-oxooxazolidin-5-yl)methyl)acetamide),preferably a Gram negative bacteria or Mycobacterium abscessus,comprising: providing a sample comprising a bacteria not sensitive to(N-(((S)-3-(dibenzo[b,e][1,4]dioxin-7-yl)-2-oxooxazolidin-5-yl)methyl)acetamide)and Mycobacterium tuberculosis or a Gram positive bacteria that issensitive to(N-(((S)-3-(dibenzo[b,e][1,4]dioxin-7-yl)-2-oxooxazolidin-5-yl)methyl)acetamide);and applying an effective amount of(N-(((S)-3-(dibenzo[b,e][1,4]dioxin-7-yl)-2-oxooxazolidin-5-yl)methyl)acetamide)to the sample to inhibit the growth of the Mycobacterium tuberculosis orthe Gram positive bacteria. The term “sensitive” means a bacteria havinga minimum inhibitory concentration of 30 or less, preferably 20 or less,preferably 10 or less, and most preferably 5 or less, when treated with(N-(((S)-3-(dibenzo[b,e][1,4]dioxin-7-yl)-2-oxooxazolidin-5-yl)methyl)acetamide)is Mycobacterium abscessius. The term “not sensitive” means a bacteriahaving a minimum inhibitory concentration of greater than 5, preferably10, preferably greater than 20, and most preferably greater than 30.

DETAILED DESCRIPTION OF THE INVENTION

T145 Inhibits Growth of Enterococcus faecalis, Staphylococcus Aureus andMycobacterium tuberculosis

The antimicrobial potency of T145 was assessed against gram negativesKlebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosaand Enterobacter cloacae, gram positives Enterococcus faecalis, andmethicillin-sensitive and -resistant Staphylococcus aureus, and acidfast pathogens Mycobacterium abscessus, Mycobacterium avium andMycobacterium tuberculosis by determining minimum inhibitoryconcentration (MIC) and minimum bactericidal concentration (MBC).Linezolid, an oxazolidinone that is clinically used for treatment ofbacterial infections, was used as a control. Meropenem, one of the mostpotent antimicrobial available in clinic today, was also included as anadditional control in this study.

T145 exhibited potent activity against growth of Mycobacteriumtuberculosis and gram positive pathogens Staphylococcus aureus andEnterococcus faecalis (Table 1). Both methicillin-sensitive and-resistant strains of Staphylococcus aureus (strains ATCC 29213 and ATCC43300) were equally sensitive to T145 with a MIC₉₀ of 0.5-1.0 μg/ml.T145 displayed no activity against the gram negative pathogensEnterobacter cloacae, Klebsiella pneumoniae and Pseudomonas aeruginosaand non-tuberculosis mycobacteria Mycobacterium abscessus andMycobacterium avium. The discovery that Mycobacterium tuberculosis wassensitive to T145 was surprising since T145 was thought to be apromising antimicrobial agent in the fight against resistantGram-positive bacterial infections but not Gram-negative bacteria suchas E. coli or acid fast bacteria such as Mycobacterium tuberculosis. Asshown in Table 1, at least one Gram negative bacteria, Acinetobacterbaumannii, was sensitive to T145, demonstrating the determination ofwhether a bacteria will be sensitive to T145 based on its Gram negativeor Gram positive phenotype is unpredictable. Mycobacterium tuberculosisis an acid fast bacteria and does not belong to either Gram negative orGram positive groups. Mycobacterium abscessus and Mycobacterium aviumwere not sensitive to T145, but Mycobacterium tuberculosis was sensitiveto T145, demonstrating the effect of T145 on bacteria, specificallyMycobacterium is unpredictable (Table 1).

Three axenic isolates of Acinetobacter baumannii were evaluated fortheir susceptibilities against T145. Acinetobacter baumannii 6M-1b is anenvironmental strain isolated from Monocacy River, Md.; 19606 is an ATCCtyped strain and ACBA is a recent clinical strain isolated from apatient at the Johns Hopkins Hospital, Baltimore, who had been treatedwith a range of antibiotics. The environmental strain 6M-1b displayedsensitivity to T145, linezolid and meropenem with a MIC₉₀ of 0.25-4.0(Table 1). Next, strain ATCC 19606 was marginally susceptible to T145only (MIC₉₀=32-64 μg/ml). With an MIC₉₀ of >64 μg/ml for T145, linezolidand meropenem, the patient isolate ACBA was most resistant among thethree strains. The environmental strain of Acinetobacter baumanniilikely was not exposed to oxazolidinones in nature and consequently wasnever under selective pressure of this agent. The patient isolate ACBAis likely to have the most exposure to drugs amongst the three strains.This data suggests that an increasing exposure to drugs has allowedAcinetobacter baumannii to develop considerable resistance to evolvedoxazolidinones T145.

It is noteworthy that potency of T145 is several fold superior tomeropenem against Enterococcus faecalis and Mycobacterium tuberculosis.The 0.5-1.0 μg/ml MIC₉₀ of T145 against Mycobacterium tuberculosis iscomparable to 0.5-2.0 μg/ml MIC₉₀ of linezolid, a clinically usedoxazolidinone. Today linezolid is one of the second line drugs used fortreatment of multi- and extensively-resistant tuberculosis based on itsin vivo potency to treat Mycobacterium tuberculosis infections. Clinicalutility of T145 will be largely determined by its pharmacokinetic,pharmacodynamics and toxicity characteristics.

TABLE 1 Minimum inhibitory concentrations (MIC₉₀) of T145 and T197 inμg/ml. Data shown in this table were verified with two repeats of MICdetermination. Meropenem and linezolid were used as - controls, ND = notdone. MIC₉₀ (μg/ml) Organism T145 Linezolid Meropenem Gram negativesAcinetobacter baumannii 6M-1b 0.25-0.50 0.25-0.50 1-2 Acinetobacterbaumannii ATCC 32-64 >64 >64 19606 Acinetobacter baumannii ACBA >64 >64 >64 Enterobacter cloacae >64 >64 0.13-0.25 Klebsiellapneumoniae >64 16-32    <0.06 Pseudomonas aeruginosa >64 >64 0.25-0.50Gram positives Staphylococcus aureus methicillin-sensitive 0.5-1.0 1-20.06-0.13 Enterococcus faecalis 0.25-0.50 1-2 4-8 MycobacteriaMycobacterium tuberculosis 0.5-1.0 0.2-2.0 ^(4, 11) 4-8 Mycobacteriumabscessus >64 ND >64 Mycobacterium avium >64 ND >64T145 is Bactericidal Against Mycobacterium tuberculosis butBacteriostatic Against Enterococcus faecalis and Staphylococcus aureus

To investigate the antimicrobial mechanism of T145 the inventorsdetermined minimum bactericidal concentration (MBC_(99.9)), theconcentration of drug at which 99.9% of bacilli are killed, forMycobacterium tuberculosis, Enterococcus faecalis and Staphylococcusaureus. For this, each pathogen was incubated in the presence of T145 at1, 2, 4 and 8 times respective MIC₉₀ and the number of surviving bacilliwere enumerated from observed colony forming units. T145 exhibitedbactericidal activity against Mycobacterium tuberculosis with MIC_(99.9)of 2 μg/ml. Enterococcus faecalis and Staphylococcus aureus grewconfluently on solid medium containing up to 8 times the MIC₉₀ of T145.Based on the lack of observable difference in CFU at and up to 8 timesMIC₉₀, the conclusion was that T145 is bacteriostatic againstEnterococcus faecalis and Staphylococcus aureus. Linezolid also exhibitsbacteriostatic activity against Staphylococcus aureus.

Frequency of Spontaneous Mutation Conferring Resistance

As a therapeutic agent, a drug becomes useless for treatment ofinfections with strains that develop resistance for it. Therefore, thefrequency at which spontaneous genetic mutation(s) that render apathogen resistant to a drug arise is a measure of the longevity of thedrug. Mycobacterium tuberculosis, Enterococcus faecalis andStaphylococcus aureus were subjected to T145 at 5×-60×MIC₉₀ to determinethe frequency of spontaneous resistant mutants. The inventors were ableto isolate Mycobacterium tuberculosis mutants after 5 weeks ofincubation at 5× and 10×MIC₉₀ of T145 and conclude that spontaneousmutants that confer resistance to T145 arise at a frequency of 4.0×10⁻⁸. Enterococcus faecalis and Staphylococcus aureus did not form classicaldistinct single colonies on agar plates containing T145. The inventorsconsistently observed a thin semi-transparent layer atop agar, which wedetermined was the initial 1 ml culture deposited on it, on every platethat contained T145 at concentrations up to 60×MIC₉₀. Repeat attempts toselect spontaneous mutants failed to produce distinct colonies. Wehypothesize that T145 is bacteriostatic against Enterococcus faecalisand Staphylococcus aureus up to 60× respective MIC₉₀ and preventsselection of spontaneous resistant mutants. To test this hypothesis, wescraped the dried semi-transparent layer and transferred to freshmedium. The recovered organisms grew consistently in liquid brothlacking T145 but failed to grow in its presence at its MIC₉₀.

Antimicrobial activity of T145 in combination with carbapenems

-   -   Carbapenems are a sub-class of 62-lactams and derive their        antimicrobial potency by inhibiting peptidoglycan biosynthesis.        The inventors hypothesized that inhibition of two essential        pathways, namely protein synthesis by oxazolidinone T145 and        peptidoglycan biosynthesis by carbapenems, may result in        synergistic killing of bacterial pathogens. To evaluate if        synergy (or indifference or antagonism) in antimicrobial        activity exists between T145 and carbapenems, the inventors used        a checkboard titration assay and subjected Enterococcus faecalis        and methicillin-sensitive and -resistant strains of        Staphylococcus aureus to combination of the two agents, each at        defined concentrations ranging from 1×MIC₉₀ to 1/16×MIC₉₀.        Parenterally administered carbapenems doripenem and biapenem, an        orally bioavailable penem faropenem and carbapenem tebipenem        were studied as representatives of this class of 62-lactams.        Both Enterococcus faecalis and Staphylococcus aureus are        sensitive to these drugs (Table 2). Growth inhibition of        pathogens tested was not observed in any wells containing less        than ½ MIC₉₀ of T145 or carbapenems demonstrating that        antibacterial activity of T145 does not synergize with the        tested carbapenems. Growth of the organisms was inhibited in        wells containing T145 at ≥½ MIC₉₀. Based on these data the        inventors conclude that antimicrobial activity of T145 is        indifferent (neither antagonistic nor synergistic) when combined        with doripenem, biapenem, faropenem and tebipenem.

The inventors also evaluated if any drug-drug interaction existedbetween T145 and isoniazid or rifampicin, two drugs that comprise thebackbone of tuberculosis treatment today, against Mycobacteriumtuberculosis using the checkerboard assay as described above. Anyantagonism or synergy between T145 and isoniazid or rifampicin is notobserved, and therefore that activity of T145 against Mycobacteriumtuberculosis is indifferent to that of isoniazid or rifampicin.

All references, including publications, patent applications, andpatents, cited herein are hereby incorporated by reference to the sameextent as if each reference were individually and specifically indicatedto be incorporated by reference and were set forth in its entiretyherein.

The use of the terms “a” and “an” and “the” and similar referents in thecontext of describing the invention (especially in the context of thefollowing claims) are to be construed to cover both the singular and theplural, unless otherwise indicated herein or clearly contradicted bycontext. The terms “comprising,” “having,” “including,” and “containing”are to be construed as open-ended terms (i.e., meaning “including, butnot limited to,”) unless otherwise noted. Recitation of ranges of valuesherein are merely intended to serve as a shorthand method of referringindividually to each separate value falling within the range, unlessotherwise indicated herein, and each separate value is incorporated intothe specification as if it were individually recited herein. All methodsdescribed herein can be performed in any suitable order unless otherwiseindicated herein or otherwise clearly contradicted by context. The useof any and all examples, or exemplary language (e.g., “such as”)provided herein, is intended merely to better illuminate the inventionand does not pose a limitation on the scope of the invention unlessotherwise claimed. No language in the specification should be construedas indicating any non-claimed element as essential to the practice ofthe invention.

Preferred embodiments of this invention are described herein, includingthe best mode known to the inventors for carrying out the invention.Variations of those preferred embodiments may become apparent to thoseof ordinary skill in the art upon reading the foregoing description. Theinventors expect skilled artisans to employ such variations asappropriate, and the inventors intend for the invention to be practicedotherwise than as specifically described herein. Accordingly, thisinvention includes all modifications and equivalents of the subjectmatter recited in the claims appended hereto as permitted by applicablelaw. Moreover, any combination of the above-described elements in allpossible variations thereof is encompassed by the invention unlessotherwise indicated herein or otherwise clearly contradicted by context.

TABLE 2 In vitro activity of doripenem, biapenem, faropenem andtebipenem against Enterococcus faecalis, methicillin- sensitiveStaphylococcus aureus and Mycobacterium tuberculosis. Minimum inhibitoryconcentrations (MIC₉₀) in μg/ml are reported here. MIC₉₀ (μg/ml)Enterococcus Staphylococcus Mycobacterium faecalis aureus tuberculosisDoripenem 0.25-0.50 0.003-0.007 2.5-5.0 ¹⁵ Biapenem 0.25-0.500.007-0.015 2.5-5.0 ¹⁵ Faropenem 0.25-0.50 0.015-0.03  2.5-5.0 ¹⁵Tebipenem 0.25-0.50 0.03-0.06 1.25-2.5 ¹⁵ 

Examples/Methods

Bacterial Strains and In Vitro Growth Conditions

Mycobacterium tuberculosis H37Rv, Mycobacterium abscessus ATCC 19977,Mycobacterium avium 104 (isolated from an adult AIDS patient in SouthernCalifornia in 1983), Acinetobacter baumannii 6M-1b (an isolate fromMonocacy River, Frederick, Md.), Acinetobacter baumannii ATCC 19606,Acinetobacter baumannii ACBA (isolated from patient at the Johns HopkinsHospital) Enterococcus faecalis ATCC 19433, methicillin-sensitiveStaphylococcus aureus ATCC 29213, Enterobacter cloacae ATCC 13047,Klebsiella pneumoniae ATCC 35657 and Pseudomonas aeruginosa PA14 wereused. Mycobacterium tuberculosis, M. avium and M. abscessus were grownin Middlebrook 7H9 broth (Difco), containing 0.5% glycerol, 10% oleicacid-albumin-dextrose-catalase (OADC) and 0.05% Tween 80 or onMiddlebrook 7H10 agar plates at 37° C. Acinetobacter baumannii,Enterococcus faecalis, Staphylococcus aureus, Enterobacter cloacae,Klebsiella pneumoniae and Pseudomonas aeruginosa were grown incation-adjusted Mueller-Hinton broth (Becton-Dickinson). Liquid cultureswere grown with constant shaking at 220 round per minute at 37° C.Meropenem, doripenem, faropenem, biapenem, tebipenem, clarithromycin andisoniazid were commercially acquired (Sigma-Aldrich). T145 wassynthesized in the laboratory to a purity of greater than 99%.

Minimum Inhibitory Concentration

Minimum inhibitory concentration (MIC₉₀)) for Mycobacterium tuberculosiswas determined using standard broth macrodilution in 15-ml sterileconical tubes containing 2.5 ml of 7H9 broth. Standard brothmicrodilution method (using 96-well plates) was used for other organismsas per Clinical and Laboratory Standard Institute recommendations.Middlebrook 7H9 broth was used for Mycobacterium abscessus,Mycobacterium avium and Mycobacterium tuberculosis and cation-adjustedMueller-Hinton broth (Becton-Dickinson) was used for other organisms asper Clinical and Laboratory Standard Institute (CLSI) guidelines. Insummary, 10⁵ bacilli grown to exponential phase in liquid medium wereinoculated into each well containing drug at two fold dilutions rangingfrom 64 μg/ml to 0.03 μg/ml. Growth medium alone and without drug butinoculated with 10⁵ bacilli were included as negative and positivecontrols, respectively. Appropriate drugs (isoniazid for Mycobacteriumtuberculosis, doripenem for Mycobacterium abscessus, clarithromycin forMycobacterium avium and meropenem for Acinetobacter baumannii,Enterococcus faecalis, Staphylococcus aureus, Enterobacter cloacae,Klebsiella pneumoniae and Pseudomonas aeruginosa) were included aspositive control for growth inhibition. Growth was evaluated by visualinspection following incubation (at 35° C. for 18 hours forAcinetobacter baumannii, Enterococcus faecalis, methicillin-sensitiveStaphylococcus aureus, Enterobacter cloacae, Klebsiella pneumoniae andPseudomonas aeruginosa, fourteen days for Mycobacterium tuberculosis at37° C., seven days at 37° C. for M. avium and at 30° C. for 3 days forMycobacterium abscessus) as per CLSI guidelines.¹⁸ MIC₉₀ is expressed asa range spanning two concentrations: the higher concentration representsthe lowest concentration at which bacterial growth could not beobserved.

Minimum Bactericidal Concentration

Minimum bactericidal concentration (MBC_(99.9)), the minimumconcentration of drug that kills 99.9% of bacilli, was determined byextending the broth microdilution assay described above. Survivingbacilli or colony forming units (CFU) in well in which growth could notbe observed were determined by growing them on agar plates containingappropriate growth medium and enumerating CFU (after 2 days ofincubation at 37° C. for Acinetobacter baumannii, Enterococcus faecalis,Staphylococcus aureus and Mycobacterium abscessus and 21 days forMycobacterium tuberculosis). The CFU at the initiation of the study,determined by enumerating live bacilli from initial inoculum on agarplates, is the input inoculum. The concentration of the agent at which99.9% of the input CFU is killed is reported here as MIC_(99.9).

Checkerboard Titration Assay

This assay is a modification of the broth dilution assay and was carriedout as described. In summary, each well containing 10⁵ CFU received twodrugs each in two fold dilutions below their respective MIC₉₀. Thesuspensions are incubated at 37° C. and growth was evaluated asdescribed above. Fractional Inhibitory Concentration (FIC) of a drug ina combination that inhibits bacterial growth is the concentration of thedrug in the well divided by MIC₉₀ of the drug if used alone. The sum ofFIC of each drug in the combination is the FIC index. In each well wherebacterial growth was absent, FIC index was calculated and an average FICindex was determined. Drug-drug antagonism was inferred if average FICindex is >2, indifference if 0.5-2.0 and synergy if ≤0.5.

Determination of Frequency of Drug Resistance Emergence

Each organism was grown as described above to exponential phase, asuspension with optical density (A_(600nm)) of 1.0 was prepared and 1.0ml of this suspension was spread onto each plate containing solid agarwith growth media that was supplemented with T145 at 4-20× respectiveMIC₉₀. Five plates were used for each concentration of T145 studied.Colonies that grew at 37° C. (>2-3 days of incubation for Staphylococcusaureus and 21 days for Mycobacterium tuberculosis) were enumerated asspontaneous mutants resistant to T145. Colonies that grew on mediumlacking T145 were enumerated as input CFU. The number of resistantmutants as a percentage of the input CFU inoculum was calculated as thefrequency of spontaneous mutants resistant to T145.

Embodiments of the disclosure concern methods and/or compositions fortreating and/or preventing a bacterial infection, such as Mycobacteriumtuberculosis, in a subject. In certain embodiments, individuals with abacterial infection such as Mycobacterium tuberculosis are treated witha chemical entity of the present invention, such as T145.

In certain embodiments, the level to which one or more chemical entitiesof the present invention inhibits the grow of a bacteria may be anylevel so long as it provides amelioration of at least one symptom of thebacterial infection. The level of bacterial growth may decrease by atleast 2, 3, 4, 5, 10, 25, 50, 100, 1000, or more fold expressioncompared to the level of expression in a standard, or when compared toperson who has a bacteria infection but is not administered a chemicalentity of the present invention, at least in some cases. An individualmay monitor the growth of bacteria using standard methods in the art. Anindividual known to have a bacterial infection or suspected of having abacterial infection may be provided an effective amount of chemicalentity of the present invention, including T145.

In particular embodiments of the disclosure, an individual is given anantibacterial agent in addition to the one or more chemical entities ofthe present invention. When combination therapy is employed with one ormore chemical entities of the present invention, the additional therapymay be given prior to, at the same time as, and/or subsequent to thechemical entity of the present invention such as T145.

Pharmaceutical Preparations

Pharmaceutical compositions of the present invention comprise aneffective amount of one or more chemical entities of the presentinvention such as T145, dissolved or dispersed in a pharmaceuticallyacceptable carrier. The phrases “pharmaceutical or pharmacologicallyacceptable” refers to molecular entities and compositions that do notproduce an adverse, allergic or other untoward reaction whenadministered to an animal, such as, for example, a human, asappropriate. The preparation of a pharmaceutical composition thatcomprises at least one chemical entity of the present invention, oradditional active ingredient, will be known to those of skill in the artin light of the present disclosure, as exemplified by Remington: TheScience and Practice of Pharmacy, 21st Ed. Lippincott Williams andWilkins, 2005, incorporated herein by reference. Moreover, for animal(e.g., human) administration, it will be understood that preparationsshould meet sterility, pyrogenicity, general safety and purity standardsas required by FDA Office of Biological Standards.

As used herein, “pharmaceutically acceptable carrier” includes any andall solvents, dispersion media, coatings, surfactants, antioxidants,preservatives (e.g., antibacterial agents, antifungal agents), isotonicagents, absorption delaying agents, salts, preservatives, drugs, drugstabilizers, gels, binders, excipients, disintegration agents,lubricants, sweetening agents, flavoring agents, dyes, such likematerials and combinations thereof, as would be known to one of ordinaryskill in the art (see, for example, Remington's Pharmaceutical Sciences,18th Ed. Mack Printing Company, 1990, pp. 1289-1329, incorporated hereinby reference). Except insofar as any conventional carrier isincompatible with the active ingredient, its use in the pharmaceuticalcompositions is contemplated.

A pharmaceutical composition including a chemical entity of the presentinvention may comprise different types of carriers depending on whetherit is to be administered in solid, liquid or aerosol form, and whetherit need to be sterile for such routes of administration as injection.The present compositions, can be administered intravenously,intradermally, transdermally, intrathecally, intraarterially,intraperitoneally, intranasally, intravaginally, intrarectally,topically, intramuscularly, subcutaneously, mucosally, orally,topically, locally, inhalation (e.g., aerosol inhalation), injection,infusion, continuous infusion, localized perfusion bathing target cellsdirectly, via a catheter, via a lavage, in cremes, in lipid compositions(e.g., liposomes), or by other method or any combination of the forgoingas would be known to one of ordinary skill in the art (see, for example,Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company,1990, incorporated herein by reference).

Pharmaceutically acceptable salts, include the acid addition salts,e.g., those formed with the free amino groups of a proteinaceouscomposition, or which are formed with inorganic acids such as forexample, hydrochloric or phosphoric acids, or such organic acids asacetic, oxalic, tartaric or mandelic acid. Salts formed with the freecarboxyl groups can also be derived from inorganic bases such as forexample, sodium, potassium, ammonium, calcium or ferric hydroxides; orsuch organic bases as isopropylamine, trimethylamine, histidine orprocaine. Upon formulation, solutions will be administered in a mannercompatible with the dosage formulation and in such amount as istherapeutically effective. The formulations are easily administered in avariety of dosage forms such as formulated for parenteraladministrations such as injectable solutions, or aerosols for deliveryto the lungs, or formulated for alimentary administrations such as drugrelease capsules and the like. Further in accordance with the presentdisclosure, the composition of the present invention suitable foradministration is provided in a pharmaceutically acceptable carrier withor without an inert diluent. The carrier should be assimilable andincludes liquid, semi-solid, i.e., pastes, or solid carriers. Exceptinsofar as any conventional media, agent, diluent or carrier isdetrimental to the recipient or to the therapeutic effectiveness of acomposition contained therein, its use in administrable composition foruse in practicing the methods of the present invention is appropriate.Examples of carriers or diluents include fats, oils, water, salinesolutions, lipids, liposomes, resins, binders, fillers and the like, orcombinations thereof. The composition may also comprise variousantioxidants to retard oxidation of one or more component. Additionally,the prevention of the action of microorganisms can be brought about bypreservatives such as various antibacterial and antifungal agents,including but not limited to parabens (e.g., methylparabens,propylparabens), chlorobutanol, phenol, sorbic acid, thimerosal orcombinations thereof. In accordance with the present invention, thecomposition is combined with the carrier in any convenient and practicalmanner, i.e., by solution, suspension, emulsification, admixture,encapsulation, absorption and the like. Such procedures are routine forthose skilled in the art.

In a specific embodiment of the present invention, the composition iscombined or mixed thoroughly with a semi-solid or solid carrier. Themixing can be carried out in any convenient manner such as grinding.Stabilizing agents can be also added in the mixing process in order toprotect the composition from loss of therapeutic activity, i.e.,denaturation in the stomach. Examples of stabilizers for use in an thecomposition include buffers, amino acids such as glycine and lysine,carbohydrates such as dextrose, mannose, galactose, fructose, lactose,sucrose, maltose, sorbitol, mannitol, etc.

In further embodiments, the present invention may concern the use of apharmaceutical lipid vehicle compositions that include a chemical entityof the present invention, one or more lipids, and an aqueous solvent. Asused herein, the term “lipid” will be defined to include any of a broadrange of substances that is characteristically insoluble in water andextractable with an organic solvent. This broad class of compounds arewell known to those of skill in the art, and as the term “lipid” is usedherein, it is not limited to any particular structure. Examples includecompounds which contain long-chain aliphatic hydrocarbons and theirderivatives. A lipid may be naturally occurring or synthetic (i.e.,designed or produced by man). However, a lipid is usually a biologicalsubstance. Biological lipids are well known in the art, and include forexample, neutral fats, phospholipids, phosphoglycerides, steroids,terpenes, lysolipids, glycosphingolipids, glycolipids, sulphatides,lipids with ether and ester-linked fatty acids and polymerizable lipids,and combinations thereof. Of course, compounds other than thosespecifically described herein that are understood by one of skill in theart as lipids are also encompassed by the compositions and methods ofthe present invention.

One of ordinary skill in the art would be familiar with the range oftechniques that can be employed for dispersing a composition in a lipidvehicle. For example, the antibacterial chemical entities of the presentinventions (or compositions) may be dispersed in a solution containing alipid, dissolved with a lipid, emulsified with a lipid, mixed with alipid, combined with a lipid, covalently bonded to a lipid, contained asa suspension in a lipid, contained or complexed with a micelle orliposome, or otherwise associated with a lipid or lipid structure by anymeans known to those of ordinary skill in the art. The dispersion may ormay not result in the formation of liposomes.

The actual dosage amount of a composition of the present inventionadministered to an animal patient can be determined by physical andphysiological factors such as body weight, severity of condition, thetype of disease being treated, previous or concurrent therapeuticinterventions, idiopathy of the patient and on the route ofadministration. Depending upon the dosage and the route ofadministration, the number of administrations of a preferred dosageand/or an effective amount may vary according to the response of thesubject. The practitioner responsible for administration will, in anyevent, determine the concentration of active ingredient(s) in acomposition and appropriate dose(s) for the individual subject.

In certain embodiments, pharmaceutical compositions may comprise, forexample, at least about 0.1% of an active compound. In otherembodiments, the an active compound may comprise between about 2% toabout 75% of the weight of the unit, or between about 25% to about 60%,for example, and any range derivable therein. Naturally, the amount ofactive compound(s) in each therapeutically useful composition may beprepared is such a way that a suitable dosage will be obtained in anygiven unit dose of the compound. Factors such as solubility,bioavailability, biological half-life, route of administration, productshelf life, as well as other pharmacological considerations will becontemplated by one skilled in the art of preparing such pharmaceuticalformulations, and as such, a variety of dosages and treatment regimensmay be desirable.

In other non-limiting examples, a dose may also comprise from about 1microgram/kg/body weight, about 5 microgram/kg/body weight, about 10microgram/kg/body weight, about 50 microgram/kg/body weight, about 100microgram/kg/body weight, about 200 microgram/kg/body weight, about 350microgram/kg/body weight, about 500 microgram/kg/body weight, about 1milligram/kg/body weight, about 5 milligram/kg/body weight, about 10milligram/kg/body weight, about 50 milligram/kg/body weight, about 100milligram/kg/body weight, about 200 milligram/kg/body weight, about 350milligram/kg/body weight, about 500 milligram/kg/body weight, to about1000 mg/kg/body weight or more per administration, and any rangederivable therein. In non-limiting examples of a derivable range fromthe numbers listed herein, a range of about 5 mg/kg/body weight to about100 mg/kg/body weight, about 5 microgram/kg/body weight to about 500milligram/kg/body weight, etc., can be administered, based on thenumbers described above.

Alimentary Compositions and Formulations

In one embodiment of the present disclosure, the antibacterial chemicalentities of the present invention are formulated to be administered viaan alimentary route. Alimentary routes include all possible routes ofadministration in which the composition is in direct contact with thealimentary tract. Specifically, the pharmaceutical compositionsdisclosed herein may be administered orally, buccally, rectally, orsublingually. As such, these compositions may be formulated with aninert diluent or with an assimilable edible carrier, or they may beenclosed in hard- or soft-shell gelatin capsule, or they may becompressed into tablets, or they may be incorporated directly with thefood of the diet.

In certain embodiments, the active compounds may be incorporated withexcipients and used in the form of ingestible tablets, buccal tables,troches, capsules, elixirs, suspensions, syrups, wafers, and the like(Mathiowitz et al., 1997; Hwang et al., 1998; U.S. Pat. Nos. 5,641,515;5,580,579 and 5,792, 451, each specifically incorporated herein byreference in its entirety). The tablets, troches, pills, capsules andthe like may also contain the following: a binder, such as, for example,gum tragacanth, acacia, cornstarch, gelatin or combinations thereof; anexcipient, such as, for example, dicalcium phosphate, mannitol, lactose,starch, magnesium stearate, sodium saccharine, cellulose, magnesiumcarbonate or combinations thereof; a disintegrating agent, such as, forexample, corn starch, potato starch, alginic acid or combinationsthereof; a lubricant, such as, for example, magnesium stearate; asweetening agent, such as, for example, sucrose, lactose, saccharin orcombinations thereof; a flavoring agent, such as, for examplepeppermint, oil of wintergreen, cherry flavoring, orange flavoring, etc.When the dosage unit form is a capsule, it may contain, in addition tomaterials of the above type, a liquid carrier. Various other materialsmay be present as coatings or to otherwise modify the physical form ofthe dosage unit. For instance, tablets, pills, or capsules may be coatedwith shellac, sugar, or both. When the dosage form is a capsule, it maycontain, in addition to materials of the above type, carriers such as aliquid carrier. Gelatin capsules, tablets, or pills may be entericallycoated. Enteric coatings prevent denaturation of the composition in thestomach or upper bowel where the pH is acidic. See, e.g., U.S. Pat. No.5,629,001. Upon reaching the small intestines, the basic pH thereindissolves the coating and permits the composition to be released andabsorbed by specialized cells, e.g., epithelial enterocytes and Peyer'spatch M cells. A syrup of elixir may contain the active compound sucroseas a sweetening agent methyl and propylparabens as preservatives, a dyeand flavoring, such as cherry or orange flavor. Of course, any materialused in preparing any dosage unit form should be pharmaceutically pureand substantially non-toxic in the amounts employed. In addition, theactive compounds may be incorporated into sustained-release preparationand formulations.

For oral administration the compositions of the present disclosure mayalternatively be incorporated with one or more excipients in the form ofa mouthwash, dentifrice, buccal tablet, oral spray, or sublingualorally-administered formulation. For example, a mouthwash may beprepared incorporating the active ingredient in the required amount inan appropriate solvent, such as a sodium borate solution (Dobell'sSolution). Alternatively, the active ingredient may be incorporated intoan oral solution such as one containing sodium borate, glycerin andpotassium bicarbonate, or dispersed in a dentifrice, or added in atherapeutically-effective amount to a composition that may includewater, binders, abrasives, flavoring agents, foaming agents, andhumectants. Alternatively the compositions may be fashioned into atablet or solution form that may be placed under the tongue or otherwisedissolved in the mouth.

Additional formulations which are suitable for other modes of alimentaryadministration include suppositories. Suppositories are solid dosageforms of various weights and shapes, usually medicated, for insertioninto the rectum. After insertion, suppositories soften, melt or dissolvein the cavity fluids. In general, for suppositories, traditionalcarriers may include, for example, polyalkylene glycols, triglyceridesor combinations thereof. In certain embodiments, suppositories may beformed from mixtures containing, for example, the active ingredient inthe range of about 0.5% to about 10%, and preferably about 1% to about2%.

Parenteral Compositions and Formulations

In further embodiments, one or more chemical entities of the presentinvention may be administered via a parenteral route. As used herein,the term “parenteral” includes routes that bypass the alimentary tract.Specifically, the pharmaceutical compositions disclosed herein may beadministered for example, but not limited to intravenously,intradermally, intramuscularly, intraarterially, intrathecally,subcutaneous, or intraperitoneally U.S. Pat. Nos. 6,7537,514, 6,613,308,5,466,468, 5,543,158; 5,641,515; and 5,399,363 (each specificallyincorporated herein by reference in its entirety).

Solutions of the active compounds as free base or pharmacologicallyacceptable salts may be prepared in water suitably mixed with asurfactant, such as hydroxypropylcellulose. Dispersions may also beprepared in glycerol, liquid polyethylene glycols, and mixtures thereofand in oils. Under ordinary conditions of storage and use, thesepreparations contain a preservative to prevent the growth ofmicroorganisms. The pharmaceutical forms suitable for injectable useinclude sterile aqueous solutions or dispersions and sterile powders forthe extemporaneous preparation of sterile injectable solutions ordispersions (U.S. Pat. No. 5,466,468, specifically incorporated hereinby reference in its entirety). In all cases the form must be sterile andmust be fluid to the extent that easy injectability exists. It must bestable under the conditions of manufacture and storage and must bepreserved against the contaminating action of microorganisms, such asbacteria and fungi. The carrier can be a solvent or dispersion mediumcontaining, for example, water, ethanol, polyol (i.e., glycerol,propylene glycol, and liquid polyethylene glycol, and the like),suitable mixtures thereof, and/or vegetable oils. Proper fluidity may bemaintained, for example, by the use of a coating, such as lecithin, bythe maintenance of the required particle size in the case of dispersionand by the use of surfactants. The prevention of the action ofmicroorganisms can be brought about by various antibacterial andantifungal agents, for example, parabens, chlorobutanol, phenol, sorbicacid, thimerosal, and the like. In many cases, it will be preferable toinclude isotonic agents, for example, sugars or sodium chloride.Prolonged absorption of the injectable compositions can be brought aboutby the use in the compositions of agents delaying absorption, forexample, aluminum monostearate and gelatin.

For parenteral administration in an aqueous solution, for example, thesolution should be suitably buffered if necessary and the liquid diluentfirst rendered isotonic with sufficient saline or glucose. Theseparticular aqueous solutions are especially suitable for intravenous,intramuscular, subcutaneous, and intraperitoneal administration. In thisconnection, sterile aqueous media that can be employed will be known tothose of skill in the art in light of the present disclosure. Forexample, one dosage may be dissolved in isotonic NaCl solution andeither added hypodermoclysis fluid or injected at the proposed site ofinfusion, (see for example, “Remington's Pharmaceutical Sciences” 15thEdition, pages 1035-1038 and 1570-1580). Some variation in dosage willnecessarily occur depending on the condition of the subject beingtreated. The person responsible for administration will, in any event,determine the appropriate dose for the individual subject. Moreover, forhuman administration, preparations should meet sterility, pyrogenicity,general safety and purity standards as required by FDA Office ofBiologics standards.

Sterile injectable solutions are prepared by incorporating the activecompounds in the required amount in the appropriate solvent with severalof the other ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the various sterilized active ingredients into a sterilevehicle which contains the basic dispersion medium and the requiredother ingredients from those enumerated above. In the case of sterilepowders for the preparation of sterile injectable solutions, thepreferred methods of preparation are vacuum-drying and freeze-dryingtechniques which yield a powder of the active ingredient plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof. A powdered composition is combined with a liquidcarrier such as, e.g., water or a saline solution, with or without astabilizing agent.

Miscellaneous Pharmaceutical Compositions and Formulations

In other preferred embodiments of the invention, the activeantibacterial compound of the present invention, such as T145, may beformulated for administration via various miscellaneous routes, forexample, topical (i.e., transdermal) administration, mucosaladministration (intranasal, vaginal, etc.) and/or inhalation.Pharmaceutical compositions for topical administration may include theactive compound formulated for a medicated application such as anointment, paste, cream or powder. Ointments include all oleaginous,adsorption, emulsion and water-solubly based compositions for topicalapplication, while creams and lotions are those compositions thatinclude an emulsion base only. Topically administered medications maycontain a penetration enhancer to facilitate adsorption of the activeingredients through the skin. Suitable penetration enhancers includeglycerin, alcohols, alkyl methyl sulfoxides, pyrrolidones andluarocapram. Possible bases for compositions for topical applicationinclude polyethylene glycol, lanolin, cold cream and petrolatum as wellas any other suitable absorption, emulsion or water-soluble ointmentbase. Topical preparations may also include emulsifiers, gelling agents,and antimicrobial preservatives as necessary to preserve the activeingredient and provide for a homogenous mixture. Transdermaladministration of the present invention may also comprise the use of a“patch”. For example, the patch may supply one or more active substancesat a predetermined rate and in a continuous manner over a fixed periodof time.

In certain embodiments, the pharmaceutical compositions may be deliveredby eye drops, intranasal sprays, inhalation, and/or other aerosoldelivery vehicles. Methods for delivering compositions directly to thelungs via nasal aerosol sprays has been described e.g., in U.S. Pat.Nos. 5,756,353 and 5,804,212 (each specifically incorporated herein byreference in its entirety). Likewise, the delivery of drugs usingintranasal microparticle resins (Takenaga et al., 1998) andlysophosphatidyl-glycerol compounds (U.S. Pat. No. 5,725,871,specifically incorporated herein by reference in its entirety) are alsowell-known in the pharmaceutical arts. Likewise, transmucosal drugdelivery in the form of a polytetrafluoroetheylene support matrix isdescribed in U.S. Pat. No. 5,780,045 (specifically incorporated hereinby reference in its entirety).

The term aerosol refers to a colloidal system of finely divided solid ofliquid particles dispersed in a liquefied or pressurized gas propellant.The typical aerosol of the present invention for inhalation will consistof a suspension of active ingredients in liquid propellant or a mixtureof liquid propellant and a suitable solvent. Suitable propellantsinclude hydrocarbons and hydrocarbon ethers. Suitable containers willvary according to the pressure requirements of the propellant.Administration of the aerosol will vary according to subject's age,weight and the severity and response of the symptoms.

Kits of the Disclosure

Any of the compositions and/or chemical entities described herein may becomprised in a kit. In a non-limiting example, an antibacterial chemicalentity of the present invention (for example, T145) may be comprised ina kit.

The kits may comprise a suitably aliquoted inducer of antibacterialchemical entities of the present invention and, in some cases, one ormore additional agents. The component(s) of the kits may be packagedeither in aqueous media or in lyophilized form. The container means ofthe kits will generally include at least one vial, test tube, flask,bottle, syringe or other container means, into which a component may beplaced, and preferably, suitably aliquoted. Where there are more thanone component in the kit, the kit also will generally contain a second,third or other additional container into which the additional componentsmay be separately placed. However, various combinations of componentsmay be comprised in a vial. The kits of the present invention also willtypically include a means for containing the one or more chemicalentities of the present invention and any other reagent containers inclose confinement for commercial sale. Such containers may includeinjection or blow-molded plastic containers into which the desired vialsare retained.

When the components of the kit are provided in one and/or more liquidsolutions, the liquid solution is an aqueous solution, with a sterileaqueous solution being particularly preferred. The composition(s) of thepresent invention may be formulated into a syringeable composition. Inwhich case, the container means may itself be a syringe, pipette, and/orother such like apparatus, from which the formulation may be applied toan infected area of the body, injected into an animal, and/or evenapplied to and/or mixed with the other components of the kit. However,the components of the kit may be provided as dried powder(s). Whenreagents and/or components are provided as a dry powder, the powder canbe reconstituted by the addition of a suitable solvent. It is envisionedthat the solvent may also be provided in another container means.

The invention claimed is:
 1. A method of treating or preventingMicobacterium tuberculosis infection in a subject in need thereof,comprising administering to the subject an effective amount of(N-(((S)-3-(dibenzo[b,e][1,4]dioxin-7-yl)-2-oxooxazolidin-5-yl)methyl)acetamide)or a pharmaceutically acceptable salt, solvate, or stereoisomer.
 2. Themethod of claim 1 wherein the subject is a human.
 3. The method of claim1 wherein the subject is an animal.
 4. The method of claim 1 furtheradministering at least one or more antimicrobial compounds.
 5. A methodfor the selection and isolation of a bacteria not sensitive to(N-(((S)-3-(dibenzo[b,e][1,4]dioxin-7-yl)-2-oxooxazolidin-5-yl)methyl)acetamide)comprising: a. providing a sample comprising a bacteria not sensitive to(N-(((S)-3-(dibenzo[b,e][1,4]dioxin-7-yl)-2-oxooxazolidin-5-yl)methyl)acetamide) andMycobacterium tuberculosis or a Gram positive bacteria that is sensitiveto (N-(((S)-3-(dibenzo[b,e][1,4]dioxin-7-yl)2-oxooxazolidin-5-yl)methyl)acetamide); and b. applying an effective amount of(N-(((S)-3-(dibenzo[b,e][1,4]dioxin-7-yl)-2-oxooxazolidin-5-methyl)acetamide)to the sample to inhibit the growth of the Mycobacterium tuberculosis orthe Gram positive bacteria.
 6. The method of claim 5 where the bacterianot sensitive to (N-(((S)-3-(dibenzo[b,e][1,4]dioxin-7-yl)-2-oxooxazolidin-5-yl)methyl)acetamide) isMycobacterium abscessius.
 7. The method of claim 5 wherein not sensitiveis a bacteria having a minimum inhibitory concentration of 30 or greaterwhen treated with (N-q(((S)-3-(dibenzo[b,e][1,4]dioxin-7-yl)-2-oxooxazolidin-5-yl)methyl)acetamide) isMycobacterium abscessius.
 8. The method of claim 5 wherein sensitive isa bacteria having a minimum inhibitory concentration of 5 or less whentreated with (N-(((S)-3-(dibenzo[b,e][1,4]dioxin-7-yl)-2-oxooxazolidin-5-yl)methyl)acetamide) isMycobacterium abscessius.